Sickling is thought to play a major role in complications such as anemia and vaso-occlusive painful crises in SCD. An important factor for these complications may be an increased cell rigidity, mainly cause by polymer formation of deoxy Hb S and cell dehydration. Recent studies suggested that reticulocytes, young red blood cells, may play a key role in clinical course since reticulocytes are more susceptible to dehydration that mature erythrocytes. However, the mechanism of cell dehydration does not seem to be so simple. A preliminary study showed at least two populations of reticulocytes regarding sickling tendency; one subset was more susceptible to sickling than others, while erythrocytes were a single population and less susceptible to sickling. This suggests that the high susceptibility of reticulocytes to sickling and dehydration may be lost or reduced during maturation or the reticulocytes susceptible to sickling may be excluded from circulation before maturation. In this proposal, we will focus on differences in the rates of sickling and dehydration among reticulocytes of different levels of maturation. (Specific Aim 1) We will study the relationship between the rates of sickling and cell dehydration and maturation levels of SS cells. Using fluorescence image cytometry, the maturation levels will be objectively assessed by quantitating RNA amount or identifying the makers to indicate the levels. Our hypothesis is that younger reticulocytes are more susceptible to dehydration and sickling under hypoxic conditions. Specific aim 2 deals with the estimation of critical levels of Hb F required to inhibit sickling and cell dehydration for SS cells of different maturation levels. The hypothesis is that younger reticulocytes need higher Hb F levels since these cells may be more susceptible to dehydration. (Specific aim 3) To relate with clinical severity, we will investigate the in vivo sickling of SS cells obtained from the venous circulation. Our hypothesis is that a greater proportion of sickled cells may be reticulocytes. The relationship between percent sickled reticulocytes and clinical severity will be studied. (Specific aim 4) Hydroxyurea (HU) is being examined to treat SCD patients because this agent increase Hb F levels. Our hypothesis is that HU has additional effects on maturation levels of SS reticulocytes, and therefore the rates of sickling and dehydration of SS cells from patients on HU are reduced even when their Hb F levels are not raised. We will test this hypothesis by examining maturation levels of reticulocytes before and after the HU treatment and correlate their maturation levels with the rates of sickling and dehydration of these cells in vitro. We will also examine the direct effects of HU on in vitro sickling and dehydration. The combined effects of HU with know ion transport inhibitors will also be studied. We believe these studies provide will us with important information for the treatment of SCD patients.